Correlation Studies On Breast Cancer And Virus By PCR-MS And The Second Generation Sequencing Technology

Background:Breast cancer is the most common type of malignant tumor in women all over theworld.It is estimated that on a global scale there are about1.38million new cases andmore than45million people die from this cancer per year.It is known that breast cancer isa multi-factorial disease, including various risk factors, such as genetic,hormonic andenvironmental factors. In recent studies, nearly5%-10%of breast cancer cases are causedby gene mutations, however, the aetiology of most breast cancer cases remains unknown.Infections with certain viruses, bacteria and parasites have been recognized as the majorcauses of cancer and have been reported to be associated with16%of human cancers.Therefore, vaccination may be an effective way of preventing the onset of these cancers. According to the report of International Agency for research on Cancer (IARC),Epstein-Barr virus (EBV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Kaposi’ssarcoma herpes virus (KSHV), Human immunodeficiency virus type1(HIV-1), high-riskHuman papillomavirus (HPV), Human T-cell lymphotrophic virus type-1(HTLV-1),Helicobacter pylori, Clonorchis sinensis, Opisthorchis viverrini and Schistosomahaematobium are classified into the microorganisims which are carcinogenic. For example,chronically infected with hepatitis B virus or hepatitis C virus will lead to the occurance ofhepatocellular carcinoma finally. Chronic infection with HCV can also cause non-Hodgkinlymphoma, especially the B-cell lymphoma. These findings provide a valuable idea for theprevention and treatment of cancers.The question of which viruses are related to the breast cancer had puzzled researchersfor decades since Bittner discovered that the mouse mammary tumor virus (MMTV) couldcause mammary cancers in mice. Many studies indicates a possible link between breastcancer pathogenesis and virus infections, particularly HPV, EBV, MMTV, simian virus40(SV40) and KSHV.It is controversial about the pathogenic effect of HPV infection in the studies ofbreast cancer. Some research claimed that about20%of breast cancerous tissues presentedpositive of the high risk subtype of HPV, especially HPV16,18and33. On the contrary,other researches have not detected any HPV subtypes in breast cancerous tissues. Amongthe reasons, we think the opposite results may be related to different techniques and skillsin different research stages.Objective:Our research aims to detect the presence of virus in the breast cancer samples andconsequently find possible pathogenesis of breast cancer through the newly and quicklydeveloping flight mass spectrometry and high-throughput gene sequencing technology.Methods and Results:Multiplex PCR detected by matrix-assisted laser desorption ionization–time of flight(MALDI-TOF) mass spectrometry (PCR-MS) is a powerful tool for microbial detection and confirmation, which can detect multiple infections simultaneously. Recently, we havedeveloped a new high-accuracy HPV genotyping method (MS-HPV). That is to say, wecan detect30oncogenic HPV subtypes by examining two genes (E6and L1) usingPCR-MS. This method targets only one gene (E6or L1), which can avoid the commonfalse-negative results of current mass spectrometric detection. Our PCR-MS methods canprevent PCR remaining-contamination and multi-probes cross-contamination.In this study, firstly we used PCR-MS method to detect135paraffin-fixed cervicalcancerous samples, meanwhile we examined all the samples with traditional PCR,sequencing technology and commercial HPV detection kit. Then, we compared theseresults and found that the new method to detect the simple HPV infection had the highersensitivity than the commercial kit (97%;90.4%). What’s more, in the detection ofmulti-infections, the detection rates of new method and commercial kit were55.6%and25.2%, respectively. With the redetection of PCR, the positive rate of new method was upto87%(65/75).We used MS-PCR method to detect the presence of HPV, SV40，BKPyV，JCPyV，WUPyV，KIPyV，MCPyV，EBV and KSHV in the samples of breast cancer patients,breast benign tumor patients and healthy volunteers. All the positive results have beenverified by PCR and DNA sequencing. The results were: in the breast cancer samples, thedetection rates of EBV，MCPyV and HPV were74.0%，14%and2%, respectively; in thebreast benign tumor samples, the detection rates of EBV and MCPyV were32%and2%,respectively. These results suggested that the infection rates of EBV and MCPyV werestatistically significant in breast cancer samples compared with in the breast benign tumorsamples (EBV：74.0%vs.32.0%，p <0.0001；MCPyV：14.0%vs.2.0%，p=0.02).However, the difference of detection infections of the two viruses in peripheral blood wasnot statistically significant. Our results have demonstrated that EBV and MCPyV weremore prevalent in women with breast cancer than in women with benign breast disease,but it requires further studies about whether the two viruses could be regarded as thehigh-risk factors of breast cancer.We used NGS and high-performance biological information arithmetic to detect the viruses in the8samples of breast cancer. It is discovered that HPV16could be de tected inmost samples, while part of samples had infections of HBV. The results were: detectionrates of HPV in peripheral blood, metastatic lymph nodes, cancerous tissues andpara-carcinoma tissues were5/8，7/8，7/8，7/8, respectively; detection rates of HBV inperipheral blood, metastatic lymph nodes, cancerous tissues and para-carcinoma tissueswere7/8，8/8，7/8，8/8, respectively. Among the four kinds of tissues, HPV averageabundances ranked from low to high was peripheral blood, para-carcinoma tissues,cancerous tissues, metastatic lymph nodes and HBV average abundances ranked from lowto high was peripheral blood, metastatic lymph nodes, para-carcinoma tissues, canceroustissues. In addition, we have done the quantitative analysis of virus infection, includingabundance, degrees of coverage and depth. But the mechanisms of virus infection leadingto the occurance of breast cancer needs to be further studied.Conclusion:In summary, our studies found that virus infection is indeed present in breast cancer.However, its specific role in the pathogenesis of breast cancer remains to be furtherstudied.